For ELISA-like quantitation of 22 different circulating histone H3 modification patterns on the same plate

Histone extracts were prepared from HL-60 cells using the EpiQuik Total Histone Extraction Kit (Cat. #OP-0006) and spiked into bovine plasma at different concentrations. The signal intensity of each H3 modification was measured using the EpiQuik™ Circulating Modified Histone H3 Multiplex Assay Kit (Colorimetric).

The EpiQuik Circulating Modified Histone H3 Multiplex Assay Kit (Colorimetric) is a complete set of optimized buffers and reagents to detect and quantify up to twenty-two (22) modified histone H3 patterns simultaneously in a simple, ELISA-like format with use of a standard microplate reader. The kit has the following advantages and features:

Simultaneously measure 22 different histone H3 modifications, which include the most important and well-characterized patterns.

H3K4me1

H3K4me2

H3K4me3

H3K9me1

H3K9me2

H3K9me3

H3K27me1

H3K27me2

H3K27me3

H3K36me1

H3K36me2

H3K36me3

H3K79me1

H3K79me2

H3K79me3

H3K9ac

H3K14ac

H3K18ac

H3K27ac

H3K56ac

H3ser10P

H3cit

Total H3

Quick and efficient procedure, which can be finished within 2.5 hours.

Innovative colorimetric assay without the need for radioactivity, electrophoresis, chromatography, or expensive equipment.

High sensitivity with a detection limit as low as 0.5 ng/well for each modification pattern with dynamic range of 1-20 ng/well within the indicated amount range of the plasma/serum.

Total histone H3 sets are included, which can be used for normalizing total histone H3 levels for relative comparison of histone H3 content between different samples or different treatment conditions.

Strip microplate format makes the assay flexible: manual or high throughput, which enables analysis of a single modification or total 22 modification patterns within the same samples.

An assay standard control and two extra 8-well strips are conveniently included in the kit which can be used for signal intensity comparison between the assay control and the samples to indicate the amount of each histone H3 modification captured from the samples.

Simple, reliable, and consistent assay conditions.

Background InformationHistone modifications have been defined as epigenetic modifiers. Post-translational modifications of histones include the acetylation of specific lysine residues by histone acetyltransferases (HATs), deacetylation by histone deacetylases (HDACs), methylation of lysine and arginine residues by histone methytransferases (HMTs), the demethylation of lysine residues by histone demethylases (HDMTs), and the phosphorylation of specific serine groups by histone kinases (HKs). Additional histone modifications include the attachment of ubiquitin (Ub), small ubiquitin-like modifiers (SUMOs), and poly ADP-ribose (PAR) units. Next to DNA methylation, histone acetylation and histone methylation are the most well characterized epigenetic marks. Generally, tri-methylation at H3-K4, H3-K36, or H3-K79 results in an open chromatin configuration and is therefore characteristic of euchromatin. Euchromatin is also characterized by a high level of histone acetylation, which is mediated by histone acetyltransferases. Conversely, histone deacetylases have the ability to remove this epigenetic mark, which leads to transcriptional repression. Condensed heterochromatin is enriched in tri-methylation of H3-K9 and H3-K27 and silencing of euchromatin loci caused by histone deacetylation involves the recruitment of specific K9 histone methyltransferases. Methylated H3-K9 provides a binding site for the chromodomain-containing heterochromatin protein 1 (HP1), which induces transcriptional repression and heterochromatinization. At euchromatic loci, this process is mediated by co-repressors, such as retinoblastoma protein pRb or KAP1. Histone demethylases have the opposite effect on transcription. For example, the histone demethylase LSD1 is responsible for H3K4 demethylation, which leads to transcriptional inactivation. Other histone demethylases, such as jumonji (JHDM2A), are responsible for H3K9 demethylation, whereas JHDM1 has the ability to convert active chromatin marks such as H3-K36me2, to an unmodified state. Lysine residues can be mono-, di-, or trimethylated, each of which can differentially regulate chromatin structure and transcription. Along with other histone modifications such as phosphorylation, this enormous variation leads to a multiplicity of possible combinations of different modifications. This may constitute a “histone code”, which can be read and interpreted by different cellular factors.

Principle & ProcedureThis kit is designed for measuring multiple histone H3 modifications simultaneously from plasma and serum. In an assay with this kit, each histone H3 modified at specific sites will be captured by an antibody that is coated on the strip wells and specifically targets the appropriate histone modification pattern. The captured histone modified at specific sites will be detected with a detection antibody, followed by a color development reagent. The ratio of modified histone is proportional to the intensity of absorbance measured by a microplate reader at a wavelength of 450 nm.

Starting MaterialsInput Amount: The amount of plasma or serum for each assay can be 10 to 50 µl with an optimal amount of 30 µl.

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